The signal transducer and activator of transcription (STAT) family of transcription factors transduce signals from a variety of extracellular stimuli and are important mediators of inflammation, cell survival, differentiation, and proliferation (1, 2). STATs are activated in response to growth factors, cytokines, and G-CSF binding to cell surface receptor tyrosine kinases (1-3). In resting cells STATs are inactive in the cytoplasm. In response to stimuli, STATs are phosphorylated by the Janus-activated kinases (Jaks), which induces STAT dimerization and nuclear translocation, where STATs bind to specific enhancer elements in target genes (2). Although structurally similar, the seven STAT family member (STATs 1, 2, 3, 4, 5a, 5b, and 6) possess diverse biological roles (2). For example, STAT1 activation is pro-inflammatory and anti-proliferative, while STAT3 activation is anti-inflammatory and pro-apoptotic (2). STAT1 is largely responsible for mediating the effects of IFN-gamma, while STAT3 is predominantly involved in IL-6 signaling (4). STAT1 induces expression of genes that inhibit the cell cycle, and thus STAT1 is considered to have tumor suppressor properties (5). Studies show that STAT3 is activated in a majority of breast and prostate cancers, and that STAT3 inhibition using RNA interference or a dominant negative leads to reduced cell proliferation, survival, and wound healing (1, 4, 6). Blocking STAT3 interaction with the epidermal growth factor receptor (EGFR) using peptide aptamers has been shown to reduce tumor growth (7). Due to the diverse roles and potent phenotypes associated with STAT signaling, the identification of selective modulators of STAT1 and STAT3 activity may lead to pharmacological tools for cancer, wound healing, and inflammatory diseases.

Assay Overview:The purpose of this assay is to determine whether a subset of compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT3 activation" (PubChem AID 871) were nonselective due to activation of STAT1. The compounds selected for testing in this AID met at least the two following criteria: 1) they were declared active in AID 871; and 2) they were declared inactive in a previous set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT1 activation" (PubChem AID 932).In this assay activation of STAT1 activity was measured using a murine NIH 3T3 fibroblast cell line that stably expresses a STAT1-luciferase construct. Test compounds were screened for their ability to increase IFN-gamma-mediated STAT1::luciferase reporter activity. Cells were exposed to test compound, followed by treatment with IFN-gamma. Changes in STAT1::luciferase activity were monitored by measuring luminescence. An activator will increase IFN-gamma-mediated STAT1 transcription, thus increasing the activation of the luciferase reporter gene, and increasing luminescence. As designed, test compounds that increase STAT1 activity are considered non-selective activators.Protocol Summary:The activator and inhibitor counterscreen assays using STAT1::luciferase cells were run simultaneously. NIH 3T3 cells were grown in T-175 flasks in Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v fetal bovine serum and antibiotics (50 micrograms/mL each of penicillin and streptomycin) at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).Prior to the start of the assay, cells were resuspended at a density of 1.88 million cells/mL in phenol red-free growth medium, and filtered through a 0.7 micron filter. Next, 4 ul of cell suspension (7,520 cells per well) were dispensed into each well of 1536-well plates. The assay was started by immediately dispensing 28 nL of test compound (5.7 uM final nominal concentration) in DMSO to sample wells, or DMSO (0.6% final concentration) to High Control wells. Next, 28 nL of nifuroxazide (100 uM final concentration in DMSO) were added to a subset of control wells, to monitor that the assay was functioning properly. The plates were then incubated for 1 hour at 37 degrees C (5% CO2, 95% RH). Next, 1 ul of human recombinant IFN-gamma (final nominal EC80 concentration of 3.0 ng/mL, set as 100% activation) was dispensed to sample and High Control wells. The plates were then incubated for 4 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of SteadyLite HTS luciferase substrate at room temperature to each well, followed by incubation at room temperature for 15 minutes. Luminescence was measured on the ViewLux plate reader.The percent activation was defined using the following mathematical formula:% Activation = 100* (Test_Compound/Median_High_Control)Where:Test_Compound is defined as the luminescence value of wells containing IFN-gamma and test compound.Median_High_Control is defined as the median luminescence value of wells containing IFN-gamma and DMSO.Test compounds were assayed in triplicate at a single concentration of 5.7 uM. A mathematical algorithm was used to determine nominally activating compounds. Two values were calculated: (1) the average percent activation of all 1280 wells defined as "Median_Low_Control" (i.e. a "DMSO plate"), and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter: any compound that exhibited greater % activation than the cutoff parameter was declared active.The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed activation. Negative % activation values were assigned an activity score of zero.List of Reagents:Dulbecco's Modified Eagle's Media I (Invitrogen, part 11965-092)Dulbecco's Modified Eagle's Media, no Phenol Red (Invitrogen, part 21063-029)Fetal Bovine Serum (Hyclone, part SH30088-03)100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055).Nifuroxazide (Sigma-Aldrich, part N 2641)Recombinant human IFN-gamma (R&D Systems, part 485-MI)SteadyLite HTS Assay Kit (PerkinElmer, part 6016989)T175 Flasks (Corning, part 431080)1536-well plates (Greiner, part 789173)

Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate STAT1 or luciferase activity, and compounds that quench or enhance luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.Active compounds of this assay fall into the activity score range of 50 to 100 and inactive compounds have range of activity score from 23 to 50.